DISTRIBUTION OF F-ACTIN ELONGATION SITES IN LYSED POLYMORPHONUCLEAR LEUKOCYTES PARALLELS THE DISTRIBUTION OF ENDOGENOUS F-ACTIN

We compared, on lysed polymorphonuclear leukocytes (PMNs), the spatial distributions of sites that nucleate actin polymerization with the sp atial distribution of endogenous F-actin. Sites nucleating polymerizat ion of exogenous actin were detected by incubating lysed cells with rh odamine-labeled G-actin under polymerizing conditions. Endogenous F-ac tin was stabilized and stained by lysis of cells into fluorescein-labe led (FITC) phalloidin. We found the distributions of rhodamine and flu orescein intensities in a given cell, resting or stimulated with chemo attractant, to be similar. Thus, after lysis the number of sites able to nucleate actin polymerization is proportional to the local F-actin concentration. Quantitative fluorescence microscopic analysis also dem onstrated that (1) if cells were stimulated with chemoattractant short ly before lysis, the total fluorescence per cell of both fluorophors w ent up; (2) if peptide was diluted shortly before lysis, the endogenou s F-actin in the lamellae was dramatically reduced, but nucleation sit es persisted, giving a high rhodamine to fluorescein ratio; and (3) th ere was a small increase in the ratio of rhodamine (exogenously grown actin) to fluorescein (endogenous F-actin) in a region near the lamell ar/endoplasm border, centripetal to regions of the highest concentrati on of endogenous F-actin. The rhodamine signal appeared to be due to i n situ actin polymerization probably nucleated by existing free barbed ends, since (1) the rhodamine signal increased linearly with time wit h no detectable lag if the actin concentration was above that of the c ritical concentration of the barbed end; (2) the rhodamine signal was dramatically reduced if lysates were incubated with gelsolin-actin com plex (which stably caps barbed ends), then washed before the rhodamine G-actin was added; and (3) the number of nucleation sites at the time of lysis is similar to the number of the barbed ends of actin filamen ts determined by the kinetics of depolymerization [Cano et al., 1991]. The fact that the distribution of exogenous actin polymerization para lleled the endogenous F-actin suggests that the number of free barbed ends per F-actin is roughly constant. If all filament ends were free, or if a constant fraction of the filaments ends were free, these data would suggest that the mean filament length is roughly constant throug hout the cell. (C) 1993 Wiley-Liss, Inc.